Thymosin Beta-4 Modulates Cardiac Remodeling by Regulating ROCK1 Expression in Adult Mammals

Figures

• 

  Figure 1. RT-PCR confirmed miR139-5p upregulation in response to TB4 treatment in the hypoxic mammalian heart. (a) MicroRNA microarray screens indicate a significant increase in miR139-5p expression in response to TB4 treatment one and three days following coronary artery ligation in the infarcted core (n = 4 for each treatment/time point/area, respectively). (b,c) Real-time PCR investigations revealed a significant increase in miR139-5p expression in the remote areas three days following systemic TB4 administration (c). We did not detect significant alterations on the first day of treatment (b). Bars indicate standard deviation at 95% confidence intervals (RFU: Relative Fluorescence Unit; n = 4 treatment/time point/area, * p < 0.05).

• 

  Figure 2. TB4 downregulates ROCK1 protein levels in vivo following cardiac infarction. (a) Representative western blot images of the core and healthy remote areas utilizing ROCK1 primary antibody. Our analyses revealed ROCK1 to be significantly decreased at days one and three in the infarcted core and remote areas of the hypoxic adult mouse heart following systemic TB4 treatment when compared to PBS. (b) Bar charts represent the calculated density units normalized to the total protein input by Coomassie stain. Bars indicate standard deviation at 95% confidence intervals (n = 3/each, * p < 0.05).

• 

  Figure 3. mRNA expression of ROCK1 in TB4-treated mouse hearts. Real-time mRNA expression analyses of ROCK1 revealed a decrease in mRNA levels in the infarcted core and remote areas following one and three days of systemic TB4 treatment in the hypoxic hearts when compared to PBS-treated and sham-operated controls. Bars indicate standard deviation at 95% confidence intervals (n = 3/each) (All measurements were performed in triplicates).

• 

  Figure 4. Immunohistochemical analyses of infarcted mouse hearts three days following MI. ROCK1 protein levels decreased in response to TB4 treatment at the infarcted core (a–c), border (d–f) and remote (g–i) areas when compared to PBS-treated controls (m–u). At (a,m), arrowheads indicate ROCK1-positive cardiac muscle cells. At (a,b,d,e), white arrows point to surviving cardiomyocytes with strong TB4 positivity and low ROCK1 expression in the TB4-treated infarcted core and border regions. In panel (i) a thickening of the epicardial layer (white arrow) may be observed when compared to PBS-treated hearts (u), indicating activation of local progenitors following TB4 treatment. (g–j,s–u) Immunohistological analysis indicates increased ROCK1 positivity in the remote areas (g) when compared to PBS-treated controls (s). (j–l,v–x) High-powered microscopic images reveal a decrease of ROCK1 protein levels in the tunica adventitia of the mature cardiac vessels ((j,v,x) white arrows). Blue DAPI stain represents the nuclei of cells (e: vessel endothel; ta: tunica adventitia, sm: smooth muscle) (n = 3/each treatment).

• 

  Figure 5. Alterations of ROCK1, beta-Actin and MRTFA proteins following external TB4 treatment of HUVECs in norm- and hypoxic conditions in vitro. (a–j) Immunocytochemical analyses detected no significant alterations in ROCK1 (a,f) and MRTFA (d,i) levels and localization following TB4 treatment with only a moderate disassembly of actin filaments when stained for beta-Actin (b,g) during normoxic conditions. (k–t) Under hypoxic conditions, immunocytochemistry revealed altered ROCK1 levels (k,p) and a visible drop in actin filaments (l,q) without detectable alterations in MRTFA levels and localization (n,s) following TB4 treatment when compared to PBS. Nuclei of cells stained with DAPI (blue) (c,e,h,j,m,o,r,t) (n = 3/each).

• 

  Figure 6. Alterations in ROCK1, alpha-Actin and MRTFA protein levels following external TB4 treatment of human adult CMs in norm- and hypoxic conditions in vitro. Immunocytochemistry of normoxic (a–j) and hypoxic (k–t) adult human heart cells revealed a significant decrease in ROCK1 protein levels with a decrease in filamentous actin structures following external TB4 treatment (b,l) when compared to those cells with PBS (g,q). (d,i,n,s) Indicate a localization shift of MRTF-A in hCMs towards the nucleus following TB4 addition under both hypoxic and normoxic conditions (d,n) when compared to PBS-treated cells (i,s), respectively. Nuclei were stained with DAPI (blue) (c,e,h,j,m,o,r,t) (n = 3/each).

• 

  Figure 7. Alterations in ROCK1, alpha-Actin, alpha-smooth muscle Actin and MRTFA proteins of cultured human adult CFBs under normoxic and hypoxic conditions following TB4 treatment. Immunocytochemistry revealed a significant decrease in ROCK1 expression following TB4 treatment under both normoxic (a,i,d,l) and hypoxic (q,y,t,bb) conditions. (b,j,r,z) Panels demonstrate beta-actin filaments become disrupted following TB4 addition to the medium (b,r), and this alteration became significantly prominent under hypoxic conditions (r) when compared to PBS-treated cells (j,z). (e,m,u,cc) Panels reveal a detectable decrease in alpha-smooth muscle Actin positivity in TB4-treated hCFBs (e,u), especially under hypoxic conditions (u) when compared to PBS-treated cells (m,cc), suggesting sufficient inhibition of fibroblast-myofibroblast transformation in vitro. (g,o,w,ee) Panels show no significant alterations in MRTFA levels and localization following TB4 treatment (g,w) when compared to PBS (o,ee). Nuclei were stained with DAPI (blue) (c,f,h,k,n,p,s,v,x,aa,dd,ff) (n = 3/each).